1. Field of the Invention
The invention relates to mutants and alleles of the oxyR gene of coryneform bacteria coding for variants of the OxyR transcription regulator and methods for producing amino acids, especially selected from the group consisting of L-lysine, L-tryptophan, L-proline, L-valine, L-isoleucine and L-homoserine using bacteria which comprise these alleles.
2. Description of the Prior Art
Amino acids are used in human medicine, in the pharmaceutical industry, in the food product industry and very especially in livestock nutrition.
It is known that amino acids are produced by fermentation of strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of the great importance, work on improving the production methods is continually being done. Improvements in the methods may be fermentation technology measures such as, for example, stirring and supplying oxygen, or relate to the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by for example ion exchange chromatography or the intrinsic output properties of the microorganism itself.
The methods used for improving the output properties of these microorganisms are ones of mutagenesis, selection and choice of mutants. The strains obtained in this way are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance, and produce the amino acids. A known antimetabolite is the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC).
Methods of recombinant DNA technology have likewise been used for some years for strain improvement of L-amino acid-producing strains of Corynebacterium by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production.
The chromosome of Corynebacterium glutamicum was completely sequenced some time ago (Kalinowski et al., Journal of Biotechnology 104, 5-25 (2003)). The chromosome of Corynebacterium efficiens has likewise been sequenced (Nishio et al., Genome Res. 13 (7), 1572-1579 (2003)).
Corresponding sequence data can be taken from the public databases. Suitable databases are for example the database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK), the database of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA), that of the Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland), the Protein Information Resource Database (PIR, Washington, D.C., USA) and the DNA Data Bank of Japan (DDBJ, 1111 Yata, Mishima, 411-8540, Japan).
Summarizing descriptions of the genetics, the metabolism and the industrial importance of Corynebacterium are to be found in the articles by Ikeda, by Pfefferle et al. and by Mueller and Huebner in the book “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering 79, (2003), Springer Verlag, Berlin, Germany, editor: T. Scheper), in the special edition “A New Era in Corynebacterium glutamicum Biotechnology” of the Journal of Biotechnology (volume 104 (1-3), 2003, editors: A. Puhler and T. Tauch) and in the “Handbook of Corynebacterium glutamicum” (editors: L. Eggeling and M. Bott, CRC Press, Taylor & Francis Group, Boca Raton, Fla., USA, 2005).
The nucleotide sequence of the oxyR gene coding for the OxyR transcription regulator of Corynebacterium glutamicum is generally available inter alia in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA) under the access number AF697215. It can furthermore be found in the Patent Application EP1108790 as sequence No. 2114.
U.S. Pat. No. 6,916,636 reports the effect of overexpression of the OxyR transcription regulator on amino acid production.
For improved clarity, the nucleotide sequence of the oxyR gene coding for the OxyR transcription regulator from the wild type of Corynebacterium glutamicum (“wild-type gene”) according to the data of the NCBI database is depicted in SEQ ID NO:1, and the amino acid sequence of the encoded OxyR transcription regulator resulting therefrom is depicted in SEQ ID NO:2 and 4. Nucleotide sequences located upstream and downstream are additionally indicated in SEQ ID NO:3.